Tuberculosis (TB) kills approximately 3 million people per year. An important characteristic of M. tuberculosis is its ability to survive within human macrophages, and mycobacterial cell wall components are plausible mediators of this effect. However, little is known about the role of specific cell wall components in either mycobacterial survival within host macrophages or modulation of macrophage responses to mycobacterial infection. In our laboratory, the recent generation of a M. tuberculosis transposon-mediated mutant library provides a unique opportunity to systematically investigate these issues. Our overall objective is to understand the role of M. tuberculosis cell wall components in the pathogenesis of TB. Our hypothesis is that M. tuberculosis cell wall components modulate macrophage immune responses, and that alteration of the cell wall will affect macrophage responses to mycobacteria. The specific aim of this R03 proposal is to characterize human macrophage responses to a) infection with M. tuberculosis transposon-mediated mutants predicted to have alterations in the cell wall, and b)stimulation with cell wall fractions from those mutants. Mutants with disruptions in genes known or predicted to be involved in biosynthesis of sulfolipids, lipoarabinomannan, mycolic acids, and phthiocerol dimycocerosate will be used. Host cell cytokine production, chemokine production, apoptosis, IFN gamma-mediated responses, and toll-like receptor-mediated responses will be characterized, and mycobacterial survival and growth within macrophages will be determined. This research will lead to a better understanding of how mycobacterial cell wall components affect bacteria-host cell interactions, and will facilitate identification of new anti-tuberculosis drug targets and vaccine strategies. If cell wall components contribute to M. tuberculosis-induced immune suppression, then evaluation of appropriate mutant strains as vaccines would be warranted. These studies will also serve as a foundation for development of rapid in vitro screening systems to evaluate the effects of other M. tuberculosis genes or cellular components on targeted host immune responses. Finally, new information from the proposed self-contained pilot studies will serve as the foundation for future comprehensive dissection of macrophage response pathways affected by specific cell wall components. [unreadable] [unreadable]